Substance P derived from sensory C fibers play a major role in bladder inflammation. We presented evidence of a mandatory role of mast cells and substance P receptors in cystitis. However, it is not clear how mast cell degranulation leads to activation of sensory nerves and SP release. Using classical animal models and cDNA array technology, we characterized a common pathway that, regardless of the initiating stimulus (LPS, antigen, or SF), is activated in cystitis. This pathway includes a receptor for mast cell tryptase (protease-activating receptors; PAR), and NF-kB genes. We further presented evidence indicating that NF-kB is involved in experimental cystitis and that treatment with a proteosome inhibitor (lactacystin) or phosphodiestase 4 inhibitor (rolipram) blocks both the expression of NF-kB genes and inflammation. The central hypothesis of this proposal is that regardless of the stimulus (LPS, mast cell tryptase, or antigen-challenge of sensitized animals), bladder inflammatory responses follow a common pathway which involves:activation of mast cells, tryptase release, activation of PAR on blood vessels and sensory nerves, release of SP, activation of NK receptors, translocation of NF-kB, and translation of inflammatory genes. Therefore, therapeutic interventions to control mast cell degranulation, PAR activation, NK1 receptors activity, or NF-kB will alter the outcome and severity of cystitis. We propose to study three well-characterized models of bladder inflammation (antigen, tryptase, and LPS) in which mast cells, protease-activating receptors, and! or SP receptors have been implicated as biological modulators of the inflammatory response. The degree of bladder inflammation will be determined by a well-established morphometric analysis and cDNA arrays. Results will be confirmed by RNase protection assays and cellular patterns of expression will be performed by in situ hybridization and immunohistochemistry. Aim 1 will determine whether protease-activating receptors (PARs) play a central role in cystitis and Aim 2 will determine whether NE-kB translates the stimulus into inflammatory gene regulation. These studies will identify and define the important mechanisms involved in bladder inflammation and provide new insights for developing treatment strategies for cystitis.